The interaction of HDL apolipoproteins, particularly apoA-I, with the cell membrane transporter ABCA1 removes excess cellular cholesterol and protects against atherogenesis. This process is mediated by lipid-poor apolipoproteins generated by either de novo synthesis or dissociation from HDL particles. Thus, factors that impair the generation or lipid efflux activity of apoA-I could have profound atherogenic effects. Oxidative damage is implicated in the pathogenesis of atherosclerosis, a chronic inflammatory disease. 3-Chlorotyrosine and 3-nitrotyrosine, stable products of protein oxidation generated by phagocyte-derived hypochlorous acid (HOCI) and reactive nitrogen species (RNS), have been detected in human atherosclerotic lesions. However, the underlying factors that control tyrosine oxidation in proteins remain poorly understood. We found that oxidation of HDL-associated and free apoA-I by HOCI and peroxynitite impairs its ability to remove cellular cholesterol by the ABCA1 pathway, consistent with the possibility that oxidation of HDL apolipoproteins in vivo would be atherogenic. We propose to test the hypothesis that site-specific oxidation of tyrosine residues by phagocyte-derived HOCI and RNS alters the biological and atheroprotective function of HDL. We will characterize the effects of site-specific oxidation of tyrosines in apoA-I on its ability to remove cellular cholesterol, identify protein motifs that direct site-specific tyrosine oxidation by HOCI and RNS, and determine if HDL is a physiological target for oxidative modification in the artery wall. This project will use HPLC and tandem mass spectrometry to locate and quantify the sites of tyrosine oxidation in apoA-I and model peptides, cell biology procedures to characterize the effects of apoA-I oxidation on lipid transport activity and interactions with ABCA1 apo A-I mutagenesis to test the functional significance of site-specific modifications, tissue analysis to identify and characterize oxidized apoA-I in human atherosclerotic lesions, and mouse model approaches to test for the effects of macrophage-generated HOCI and RNS on apoA-I oxidation and atherogenesis in vivo. The proposed studies will provide insights into the structural features that direct oxidative modification of proteins, with important implications for the physiological significance of oxidative reactions in atherosclerosis and other inflammatory diseases. [unreadable] [unreadable]